Intestinal absorption of fucoidan extracted from the brown seaweed, Cladosiphon okamuranus.

The aim of this study was to examine the absorption of fucoidan through the intestinal tract. Fucoidan (0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL) was added to Transwell inserts containing Caco-2 cells. The transport of fucoidan across Caco-2 cells increased in a dose-dependent manner up to 1.0 mg/mL. It reached a maximum after 1 h and then rapidly decreased. In another experiment, rats were fed standard chow containing 2% fucoidan for one or two weeks. Immunohistochemical staining revealed that fucoidan accumulated in jejunal epithelial cells, mononuclear cells in the jejunal lamina propria and sinusoidal non-parenchymal cells in the liver. Since we previously speculated that nitrosamine may enhance the intestinal absorption of fucoidan, its absorption was estimated in rats administered N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water. Rats were fed 0.2% fucoidan chow (BBN + 0.2% fucoidan rats), 2% fucoidan chow (BBN + 2% fucoidan rats) and standard chow for eight weeks. The uptake of fucoidan through the intestinal tract seemed to be low, but was measurable by our ELISA method. Fucoidan-positive cells were abundant in the small intestinal mucosa of BBN + 2% fucoidan rats. Most fucoidan-positive cells also stained positive for ED1, suggesting that fucoidan was incorporated into intestinal macrophages. The uptake of fucoidan by Kupffer cells was observed in the livers of BBN + 2% fucoidan rats. In conclusion, the absorption of fucoidan through the small intestine was demonstrated both in vivo and in vitro.

Attenuation of N-nitrosodiethylamine-induced liver fibrosis by high-molecular-weight fucoidan derived from Cladosiphon okamuranus.

BACKGROUND AND AIM: Liver fibrosis is closely associated with the progression of various chronic liver diseases. Fucoidan exhibits different biological properties such as anti-inflammatory, anti-oxidant and anti-fibrotic activities. The aim of this study was to determine whether oral fucoidan administration inhibits N-nitrosodiethylamine (DEN)-induced liver fibrosis. METHODS: Liver fibrosis was induced in rats by injecting DEN (50 mg/kg). Rats were given 2% of crude fucoidan solution or 2% of high-molecular-weight (HMW) fucoidan solution. They were divided into a crude fucoidan group, an HMW fucoidan group, a DEN alone group, a DEN + crude fucoidan group, a DEN + HMW fucoidan group and a control group. RESULTS: Liver fibrosis and hepatic hydroxyproline levels were significantly more decreased in the DEN + HMW fucoidan group than in the DEN-alone group. Anti-fibrogenesis was unremarkable in the DEN + crude fucoidan group. Hepatic messenger RNA levels and immunohistochemistry of transforming growth factor beta 1 were markedly increased by DEN. This increase was attenuated by HMW fucoidan. Hepatic chemokine ligand 12 expression was increased by DEN. This increase was suppressed by HMW fucoidan. HMW fucoidan significantly decreased the DEN-induced malondialdehyde levels. Also, fucoidan markedly increased metallothionein expression in the liver. Fucoidan was clearly observed in the liver by immunohistochemical staining in HMW fucoidan-treated rats, while it was faintly stained in the livers of crude fucoidan-treated rats. CONCLUSION: These findings suggest that the HMW fucoidan treatment causes anti-fibrogenesis in DEN-induced liver cirrhosis through the downregulation of transforming growth factor beta 1 and chemokine ligand 12 expressions, and that scavenging lipid peroxidation is well-incorporated in the liver.

Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12.

The aim of this study is to assess whether fucoidan modulates the expression of chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) and exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC50) of 2.0 and 4.0 mg/ml, respectively. alpha-fetoprotein levels in medium collected from fucoidan-treated cells were significantly decreased in Huh7 cells but not in HepG2 cells. Western blotting revealed that the amount of alpha-fetoprotein was decreased by 1.0 mg/ml of fucoidan in Huh7 cells, whereas it was unchanged in HepG2 cells. In Huh7 cells, CXCL12 mRNA expression was significantly downregulated by 1.0 mg/ml of fucoidan, whereas CXCR4 mRNA expression was unchanged by fucoidan. CXCL12 and CXCR4 mRNA were barely expressed in HepG2 cells. In addition, 1.0 mg/ml of fucoidan mildly arrested the cell cycle and induced apoptosis in Huh7 cells. The findings suggest that fucoidan exhibits antitumor activity toward Huh7 cells through the downregulation of CXCL12 expression.

Immunohistochemical staining with newly developed metallothionein fragment antibodies against NH2-terminal, middle-regional and COOH-terminal peptides in rabbits.

Metallothionein (MT) has been reported to play important physiological roles in the human body, but the distribution and significance of MT is not fully understood. In order to analyze MT expression, three kinds of polyclonal MT fragment antibodies were developed against NH2-terminal, middle regional and COOH-terminal peptide followed by human MT-IA amino acid sequence in rabbits. The characteristics of these antibodies were studied using competitive immunoassay and immunohistochemical staining. The NH2-terminal antibody (anti-MT-N) had the strongest and clearest immunoreactivity to human and mouse tissues, while COOH-terminal antibody (anti-MT-C) showed species difference because of the 4 amino acid difference in the MT fragment peptide between human and mouse. No reactivity was detected using middle regional residue antibody (anti-MT-M) both on competitive immunoassay and on immunostaining. These results suggest that anti-MT-N is the most applicable antibody to use for immunohistochemistry in human, mouse and other specimens.

Changes of heavy metal, metallothionein and heat shock proteins in Sertoli cells induced by cadmium exposure.

In this study, we examined the levels of Cadmium (Cd), iron (Fe) and zinc (Zn), which were considered to be involved in Sertoli cell damage caused by Cd exposure. We also examined metallothionein (MT), heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1) expressions in Sertoli cells induced by Cd exposure. Evaluation by the in-air micro-particle induced X-ray emission (PIXE) method revealed that Cd and Fe distribution was increased in the cytoplasm of Sertoli cells after Cd exposure. By contrast, Zn was decreased in the cytoplasm of Sertoli cells after Cd exposure. It was suggested that the target of Cd toxicity was the cytoplasm of Sertoli cells, Fe was considered to enhance damage to Sertoli cells caused by Cd exposure. The DNA fragmentation rate was determined by ELISA after Cd exposure to Sertoli cells. It remained essentially unchanged with 2.5 microM Cd exposure of Sertoli cells; however, MT, Hsp70 and HO-1 were significantly increased by Cd exposure. As a result, Cd-induced MT was protected Sertoli cells against apoptosis, and Cd-induced HO-1 was involved in protection against oxidative stress. Incidentally, MT, Hsp70 and HO-1 showed similar responses to Cd exposure.

Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography.

INTRODUCTION: In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. METHOD: A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60×8.0mm I.D., 30nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210nm) is from 0 to 100ng of fucoidan with the linear calibration line inserting to zero. RESULTS: A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10mM sodium azide (respiration inhibitor) and 0.05mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all. CONCLUSION: We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells.

Determination of the serum metallothionein (MT)1/2 concentration in patients with Wilson's disease and Menkes disease.

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH2 terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6ng/ml and the spiked MT-1was fully recovered from the plasma. We investigated the normal range of MT1/2 (25-75%tile) in 200 healthy human serum and found it to be 27-48ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long-Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.

The anti-apoptotic effect of fucoxanthin on carbon tetrachloride-induced hepatotoxicity.

This study evaluated the anti-apoptotic activity of fucoxanthin in carbon tetrachloride (CCl(4))-induced hepatotoxicity. An in vitro study using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay clearly demonstrated an attenuation of CCl(4)-induced hepatotoxicity with fucoxanthin. This effect was dose-dependent; 25 µM was more effective than 10 µM of fucoxanthin for attenuating the hepatotoxicity induced by 5 mM of CCl(4). Acute CCl(4)-hepatotoxicity in rats, with numerous cells positive for the terminal deoxynucleotidyl - transferase (TdT) -mediated deoxyuridine triphosphate-digoxigenin (dUTP) nick-end labeling (TUNEL) stain were seen in the pericentral area of the hepatic lobule. Oral pretreatment of CCl(4)- injected rats with fucoxanthin significantly reduced hepatocyte apoptosis. Fucoxanthin was immunohistochemically shown to increase heme oxygenase-1 expression in the cultured liver cells of Hc cells and TRL1215 cells. By oral pretreatment of CCl(4)-injected rats with fucoxanthin, the hepatic heme oxygenase-1 protein levels were significantly increased compared to those not pretreated with fucoxanthin. Heme oxygenase-1 mRNA expression after CCl(4 )injection was higher in the CCl(4)+fucoxanthin group than in the CCl(4 )group, although the difference was not significant. The findings suggest that fucoxanthin attenuates hepatocyte apoptosis through heme oxygenase-1 induction in CCl(4)-induced acute liver injury.

Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals.

An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT- 3 NH(2) terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter- and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.

Metallothionein (MT) 1/2 expression in MT 1/2 and MT 3 knock-out mice and Long-Evans Cinnamon (LEC) rats.

Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies.

Development of an enzyme-linked immunosorbent assay for metallothionein-I and -II in plasma of humans and experimental animals.

BACKGROUND: An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothionein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet. METHODS: We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay. RESULTS: The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23+/-4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods. CONCLUSION: The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.

Subcellular changes of essential metal shown by in-air micro-PIXE in oral cadmium-exposed mice.

To clarify the relation of essential metals to cadmium (Cd) toxicity, we evaluated metallothionein expression and analyzed the subcellular distribution of essential metals using in-air micro-Particle-Induced X-ray Emission (PIXE). Four mice were dosed orally with 100 mg/L of Cd in drinking water for 1.5 or 2 years. Frozen samples of organs were used for micro-PIXE analysis and formalin-fixed samples were used for metallothionein staining. Immunohistochemically, metallothionein induction by 1.5y-Cd exposure was higher in the renal cortex than in the liver. Metallothionein expression was reduced after 2y-Cd administration compared to the 1.5y-Cd-exposed mice. Cd-induced tissue damage became marked in the 2y-Cd-exposed mice compared to the 1.5y-Cd-exposed mice, in which nephrotoxicity was more prominent than hepatotoxicity. Cd yield was higher in the renal cortex of the 2y-Cd-exposed mouse than in that of the 1.5y-Cd-exposed mouse, whereas no such increasing tendency was found in the liver. Compared to the control, the Cd-exposed mice markedly accumulated zinc in the liver and renal cortex. In the Cd-exposed mice, iron was mildly accumulated in the renal cortex and was slightly deprived in the liver. Elemental maps showed that a large amount of Cd was spatially combined with zinc in the 1.5y-Cd mouse. Free Cd became abundant in the 2y-Cd-exposed mouse. In addition, a small amount of Cd was colocalized with iron. The data suggest that zinc may contribute to protect against oral-administrated Cd toxicity, and impaired induction of MT may participate in hepato-nephrotoxicity of the 2y-Cd-exposed mouse.

The changes of heavy metal and metallothionein distribution in testis induced by cadmium exposure.

Cadmium (Cd) is known to cause various disorders in the testis, and metallothionein (MT) is known as a protein, which has a detoxification function for heavy metals. However, the changes of Fe, Cu, and Zn distribution in the testis induced by Cd exposure have not been well examined. Moreover, only a few studies have been reported on the localization of MT after Cd exposure. In this study, we have investigated the changes of Fe, Cu, and Zn distribution in Cd-exposed testis by a newly developed in air micro-Particle Induced X-ray Emission (PIXE) method. Also, we examined the distribution of MT expression in testis. In the testis of Cd-treated rats with significant increases of lipid peroxidation, the sertoli cell tight junction was damaged by Cd exposure, resulting from disintegration of the blood testis barrier (BTB). Evaluation by in air micro-PIXE method revealed that Cd and Fe distribution were increased in the interstitial tissues and seminiferous tubules. The histological findings indicated that the testicular tissue damage was advanced, which may have been caused by Fe flowing into seminiferous tubules followed by disintegration of the BTB. As a result, Fe was considered to enhance the tissue damage caused by Cd exposure. MT was detected in spermatogonia, spermatocytes, and Sertoli's cells in the testis of Cd-treated rats, but was not detected in interstitial tissues. These results suggested that MT was induced by Cd in spermatogonia, spermatocytes, and Sertoli's cells, and was involved in the resistance to tissue damage induced by Cd.

Role of oxidative stress on pathogenesis of hypertensive cerebrovascular lesions.

The hypertensive rat brain exhibited softening, severe edema and intracerebral hemorrhage. The NO(2) (-) + NO(3) (-) (NOx) level in the hypertensive rat brain was higher than in the normotensive rat brain. Light microscopy demonstrated severe arterial and arteriolar lesions with fibrinoid deposits and medial lesion. After injecting hypertensive rats with nitroblue tetrazolium (NBT), formazan deposits, which are the reaction product of reduction of NBT by superoxide, were observed in the microvessels and nervous tissue around the microvessels of injured brain. Immunohistochemistry showed that copper zinc superoxide dismutase and manganese superoxide dismutase expression of the endothelial cells of hypertensive rats were also upregulated in comparison with normotensive rat endothelial cells. Inducible nitric oxide synthase and endothelial nitric oxide synthase expression in endothelial cells of normotensive rats were strongly positive, whereas the expression in hypertensive rat endothelial cells was weaker. Nitrotyrosine, a biomarker of peroxynitrite, which is a powerful oxidant formed by the reaction of nitric oxide (NO) with superoxide, was found in the microvessels, injured arteries and arterioles and infarcted brain tissue. Deposition of a major aldehydic product of lipid peroxidation, that is, 4-hydroxy-2-nonenal (4-HNE) was found in microvessels, perivascular tissue, and edematous and infarcted brain. Hypertensive cerebrovascular disease is the result of hypertension-induced oxidative stress.

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells.

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.